The present invention relates to a gene involved in actin dynamics in the yeast Aspergillus fumigatus and more particularly to the identification, isolation and cloning of this gene. This invention also relates to a method of using this gene to screen for compounds with antifungal activity.
The fungal actin cytoskeleton consists of actin and diverse associated and regulatory proteins. It guides polarized growth required for extension of buds, hyphae, pseudo-hyphae and mating projections, underlies cell division, and plays a critical role in maintaining cell integrity. It is therefore essential for viability and pathogenesis. While actin is not a suitable target for anti-fungal therapeutics because fungal and human actins are 90% identical in amino acid sequence, leading to the potential cross-reactivity of inhibitors, other candidates include the numerous proteins that function in concert with actin.
Among these proteins, cofilin stands out as a particularly promising target, being 22% identical between yeast and humans, respectively. It plays a crucial role in actin regulation. More specifically, in cells actin rapidly interconverts between monomers and polymers. Only the polymer form of actin is known to be functional in vivo. Actin filaments have an inherent polarity because all subunits within the filament have the same orientation. The so-called barbed end elongates ten times more rapidly than the so-called pointed end. Each actin monomer can bind to one molecule of ATP. Hydrolysis of this ATP is slow on monomers, but is promoted by actin assembly. Significantly, at the barbed filament end ADP-actin subunits within filaments dissociate more readily from the filaments than ATP-actin subunits, which are preferred for assembly. Therefore, the consequence of assembly-stimulated hydrolysis of ATP is filament disassembly. At steady state, in the presence of excess ATP, actin subunits will very slowly flux through filaments, a process called treadmilling wherein there is net addition of ATP-actin subunits to filament barbed ends, and net loss of ADP-actin subunits from pointed ends. As a result of the balancing of these two activities, no net change in the monomer-polymer subunit distribution occurs.
While treadmilling occurs with pure actin in the presence of ATP in vitro, the rates of treadmilling are extremely slow compared to turnover rates in vivo. Cofilin and profilin greatly accelerate these rates so that the in vivo situation is approximated. Cofilin binds to actin monomers and filaments and greatly stimulates ADP-actin subunit dissociation from filaments. Molecular-genetic studies in yeast have demonstrated the importance of cofilin for promoting rapid actin dynamics; cofilin mutants show markedly reduced actin filament turnover rates.
Cofilin alone causes only a modest increase in actin treadmilling. This is because when bound to ADP-actin, cofilin inhibits nucleotide exchange. Profilin binds to actin monomers and accelerates treadmilling synergistically with cofilin because it dissociates the cofilin-ADP actin complex and promotes nucleotide exchange on actin. The resulting profilin ATP-actin complex assembles readily at filament barbed ends.
Yeast cofilin atomic structure has been solved. Moreover, extensive mutagenesis studies have assigned functions to the topological features of this protein. Significantly, the molecular-genetic studies have demonstrated critical roles for cofilin in promoting subunit disassembly and profilin in regenerating ATP-actin from cofilin-ADP-actin. Therefore, these activities have been validated as appropriate activities to target therapeutically.
Cofilin (15-19 kDa) is readily expressed in bacteria and purified. Well-established assays measure monomer and filament binding, inhibition of actin nucleotide exchange, and filament disassembly stimulation. It represents an important component of the eukaryotic cell division cycle as well as, by virtue of its interaction with the actin cytoskeleton, overall cellular integrity, vesicular transport, and cell polarity.
There is a compelling need to prevent and treat systemic fungal infections, many of which are fatal if untreated. Indeed, the 1980s and 1990s witnessed a steep rise in Candida and Aspergillus infections (Musial, C E, Cockerill III, F R, Roberts G D. (1988) Clin Microb Rev 1(4):349-364; Saral R. (1991) Reviews of Infectious Dis 13:487-492). Similar rises in zygomycosis, cryptococcosis, histoplasmosis and fusaria infection have also been noted. The reasons for the rise in fungal infections are several, but a key factor is the growing population of immuno-compromised individuals. This group includes patients with HIV disease (AIDS), older patients, patients who have undergone invasive surgery, transplant patients and burn victims.
As the population of immunosuppressed individuals increases, so do the numbers and types of fungal infections noted in these patients. Although candidiasis remains the most common fungal infection in immunosuppressed patients, aspergillosis, zygomycosis, and other infections by filamentous fungi are a major problem for an increasing number of patients (Georgiev, V. St. (1998) Infectious Diseases in Immunocompromised Hosts, CRC Press, Boca Raton, Fla.; and Fauci, A S. (1998) Emer Infect Dis. The endemic mycoses, especially histoplasmosis and coccidiodomycosis, also constitute a risk for patients. At particular risk for such infections are those with AIDS, those having undergone bone marrow or organ transplants, those receiving chemotherapy and those who have had debilitating illness, sever injury, prolonged hospitalization, or long-term treatment with antibacterial drugs (NIAID fact sheet, 1996).
According to the CDC""s National Nosocomial Surveillance System, the rate of hospital-related fungal infections nearly doubled between 1980 and 1990. In 1997, an estimated 240,000 individuals showed clinical symptoms of endemic mycoses. With the current approaches to treatment (primarily amphotericin B and the azoles) the mortality rate in patients with systemic fungal infections ranges from 30-100%, depending on the pathogen.
The severity of fungal infections increases as the immune system becomes more dysfunctional. Fungi are among the most ubiquitous pathogens seen in patients with AIDS; virtually all major fungal pathogens cause disease in HIV-positive patients. The majority of untreated HIV-positive patients experience at least one episode of fungal infection and many fungal infections are AIDS-defining illnesses in HIV-infected individuals (Phillips P. (1999).
Therefore, there is a desperate need for new antifungal agents. The recent development of high-throughput screens for the isolation of such agents presents an opportunity for meeting this need. Cofilin is amenable to such screening approaches and thus represents an important new target for antifungal drugs.
The present invention concerns an isolated nucleic acid molecule encoding A. fumigatus cofilin. Preferably, the A. fumigatus cofilin has a sequence that has greater than 70%, 80%, or 90% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm.
In one aspect, the invention provides an isolated nucleic acid sequence encoding A. fumigatus cofilin, wherein the cofilin has a sequence that has greater than 70%, 80%, or 90% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm. In one embodiment, the protein further specifically binds to polyclonal antibodies raised against SEQ ID NO:2.
In one embodiment, the nucleic acid encodes A. fumigatus cofilin, or a fragment thereof. In another embodiment, the nucleic acid encodes SEQ ID NO:2. In another embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:1.
In one aspect, the nucleic acid comprises a sequence which encodes an amino acid sequence which has greater than 70% sequence identity with SEQ ID NO:2, preferably greater than 80%, more preferably greater than 90%, more preferably greater than 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2.
In one embodiment, the nucleic acid comprises a sequence which has greater than 55 or 60% sequence identity with SEQ ID NO:1, preferably greater than 70%, more preferably greater than 80%, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:1. In another embodiment provided herein, the nucleic acid hybridizes under stringent conditions to a nucleic acid having a sequence or complementary sequence of SEQ ID NO:1.
In another aspect, the invention provides an expression vector comprising a nucleic acid encoding A. fumigatus cofilin, wherein the protein has a sequence that has greater than 70, 80, or 90% amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm. The invention further provides a host cell transfected with the vector.
In another embodiment, the protein comprises an amino acid sequence of SEQ ID NO:2. In one aspect, the protein provided herein comprises an amino acid sequence which has greater than 70% sequence identity with SEQ ID NO:2, preferably greater than 80%, more preferably greater than 90%, more preferably greater than 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2.
The invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof.
Also provided are modulators of the target protein including agents for the treatment of fungal disorders. The agents and compositions provided herein can be used in variety of applications which include the formulation of sprays, powders, and other compositions. Also provided herein are methods of treating fungal disorders.